Oligonucleotide probes and primers are a crucial component of most diagnostic assays used for detection and quantification of genomic material. For a genomic assay to be effective, it must be both highly sensitive and simultaneously produce a low false positive rate. To meet this stringent requirement, probes and primers used in an assay must be both highly sensitive and highly specific. That is, they must have a very high probability of binding to the intended sites on the target nucleic acid (e.g., DNA or RNA), but a very low probability of binding to any other background human or contaminating nucleic acid. While some current tools incorporate advanced methods to design for sensitivity, designing for specificity typically depends on BLAST (Basic Local Alignment Search Tool), which is intended for determining sequence similarity and evolutionary relatedness. Hybridization, however, is governed by sequence dependent thermodynamics, NOT by sequence similarity. Thus, BLAST is not appropriate for the task of improving hybridization stringency. For several reasons, the BLAST algorithm fails to identify many candidates for mis-hybridization, representing potential false positives. Thus there is a need to develop an algorithm that combines the efficiency of BLAST with the thermodynamic rules for hybridization. In this Fast Track SBIR application we propose development of a new software tool, ThermoBLAST, to fill this critical technology gap. This objective will be accomplished in three specific aims (aim 1 in Phase I and aims 2 and 3 in Phase II): 1) Develop prototype ThermoBLAST code modules. 2) Integrate component modules into a single application with appropriate GUI. 3) Integrate ThermoBLAST into the automated sequence design algorithms of Visual OMP. ThermoBLAST will be integrated into our OMP (Oligonucleotide Modeling Platform) software resulting in automated design of more specific probes and primers that can be designed more quickly and more economically. ThermoBLAST will allow scientists to develop better assays, while saving time and money, and thus will positively impact the quality of life of a significant segment of humanity. ThermoBLAST will make a major positive contribution to the work of public and private researchers world wide. Scientists who are designing DNA tests to identify or discover the function of important, disease related genes will be able to 'test their tests' in a computer, without having to go through the lengthy lab testing process for each assay. The most difficult DNA-based tests are also the most important. There are very small differences between the genetic code of deadly pathogens and harmless microorganisms. ThermoBLAST will greatly improve the ability of scientists to design DNA probes that yield consistent, positive results, and avoid costly (or deadly) false negative results. [unreadable] [unreadable] [unreadable]